Standard preparation— Dissolve an accurately weighed quantity of USP Methylprednisolone Acetate RSin a mixture of equal volumes of alcohol and chloroform,and dilute quantitatively with the same solvent to obtain a solution having a known concentration of about 500µg per mL.

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 5mg of methylprednisolone acetate,to a 125-mLseparator,add 50mLof solvent hexane,and mix.Extract with three 10-mLportions of acetonitrile,and evaporate the combined extracts on a steam bath with the aid of a current of air nearly to dryness.Transfer the residue to a 10-mLvolumetric flask with the aid of one 5-mLportion and two 2-mLportions of a mixture of equal volumes of alcohol and chloroform,dilute with the same solvent to volume,and mix.

Procedure— Divide the area of a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.5-mm layer of chromatographic silica gel mixture,into three equal sections,the left and right sections to be used for the Assay preparationand the Standard preparation,respectively,and the center section for the blank.Apply 250µLeach of the Assay preparationand of the Standard preparationas streaks 2.5cm from the bottom of the designated section of the plate,and dry the streaks with the aid of a current of air.Develop the chromatogram in a solvent system consisting of a mixture of ethyl acetate and chloroform (7:5)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the principal bands occupied by the Standard preparationand the Assay preparation(see also the Identificationtest)by viewing under short-wavelength UVlight.Mark these bands and the corresponding band in the section of the plate representing the blank.Quantitatively remove the silica gel containing these bands,and transfer to separate glass-stoppered,50-mLcentrifuge tubes.Add 25.0mLof alcohol to each tube,shake for 2minutes,and centrifuge at about 1500rpm for 5minutes.Transfer 20.0mLof each supernatant to separate glass-stoppered,50-mLconical flasks,add 2.0mLof blue tetrazolium TSto each solution,mix,and to each flask add 2.0mLof a mixture of 1volume of tetramethylammonium hydroxide TSand 9volumes of alcohol.Mix,and allow the solutions to stand in the dark for 90minutes.Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 525nm,with a suitable spectrophotometer,against the blank.Calculate the quantity,in mg,of methylprednisolone acetate (C24H32O6)in the portion of Cream taken by the formula: in which Cis the concentration,in µg per mL,of USP Methylprednisolone Acetate RSin the Standard preparation;and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.